Microfluidic device

ABSTRACT

Microfluidic devices of the present disclosure relate to quick and inexpensive microfluidic manipulation/handling. A number of channels may be provided for supply of fluid ingredients to a number of cavities. A separating material may provide fluid separation between a number of cavities, such as corresponding cavities of a reaction chamber. Once the cavities are supplied with fluid ingredient, channels connecting the cavities may be sealed off; that is, the cavities may be subject to fluid isolation. In some embodiments, a sealing material may be compressed so as to deform into the channels obstructing fluid flow. The separating material may be manipulated so that the initial fluid separation between cavities is removed. Removal of this fluid separation subsequently permits mixing of fluid ingredient(s) contained within previously separated cavities. In some embodiments, the fluid separation may be removed by heating or dissolving portions of the separating material. When appropriate, contents within reaction chambers may be subject to further processing (e.g., thermal cycling, various analyzes).

FIELD

Aspects of the present disclosure relate generally to methods andapparatuses for microfluidic handling.

DISCUSSION OF RELATED ART

Modern laboratory experiments often require multiple sets of reagents tobe combined according to every possible permutation. An example of suchan experiment is polymerase chain reaction (PCR). PCR typically utilizesa DNA sample, a DNA primer that complements the DNA sample at specificgene locations, and a “master mix” solution that contains DNA polymeraseand various reagents (e.g., nucleotides) that facilitate the activitiesof the polymerase.

PCR also typically involves thermal cycling. When exposed to arelatively high temperature (e.g., greater than 90 C), double helixmolecules of a DNA sample are separated into single strands. At arelatively lower temperature (e.g., 50-70 C), DNA primers attach attarget sites to single strands of the DNA sample. At an intermediaterange of temperature (e.g., 70-80 C), the polymerase facilitateselongation of DNA fragments formed from the initial attachment ofprimers to the single-stranded DNA molecules. The double-stranded DNAproducts of one PCR cycle can then be split at the relatively hightemperature range and bound to new primer strands, doubling the amountof DNA in every cycle until the reagents are exhausted. Thus, theconcentration of a DNA sample containing a target DNA sequence, whensubject to PCR, may increase exponentially.

PCR is often used for combinatorial analysis of multiple DNA samplesolutions and multiple primers. For example, ten different samples beingtested for the presence of five different genes will require fiftydifferent reactions. Because primers are often specially fabricated foreach target gene, the use of such primers can be expensive. Further,existing microfluidic systems that combine multiple groups of reagentstogether are typically complicated in design and manufacturing. As aresult, combining such reagents to test each permutation isconventionally done via hand pipetting or with a robotic system, and canbe both costly and time consuming.

Digital PCR is a type of PCR analysis that involves dividing a DNAsample into a large number of separate aliquots, and amplifying thealiquots to determine whether a molecule of target DNA was presentwithin the aliquot. Based on the number of aliquots that have undergoneexponential growth, the original concentration of DNA prior to dilutionmay be determined. Conventional systems that employ digital PCR aregenerally expensive and complex.

Thus, there is a need for simpler, less expensive microfluidic systemsthat retain the robustness of more expensive systems while minimizingthe cost per reaction.

SUMMARY

The inventors have appreciated that a microfluidic device that providesthe ability to facilitate multiple combinations of small volumereagents, in a controlled yet inexpensive manner, would be advantageous,particularly in scientific settings (e.g., PCR detection, combinatorialPCR). The inventors have further appreciated that it would be beneficialto develop a quick and cost effective system for dividing a fluidingredient evenly into a large array of separate chambers and subjectingeach of the chambers to fluid isolation from one another (e.g., fordigital PCR).

The present disclosure relates to microfluidic devices and their methodsof use and construction. Such devices may be used to accommodatemultiple series of combinatorial and/or high-throughputchemical/biological reactions more easily than existingtechniques/methods.

In an embodiment, a microfluidic device is provided. The device includesa first layer defining a first cavity; a first channel arranged toprovide fluid entry to the first cavity; a second layer defining asecond cavity; a second channel arranged to provide fluid entry to thesecond cavity; and at least one separating material providing fluidseparation between the first cavity and the second cavity, whereincompression of the first and second layers relative to one anothercauses sealing of the first channel and the second channel resulting inobstruction of the first cavity and the second cavity from further fluidentry, and wherein manipulation of the at least one separating materialcauses removal of the fluid separation allowing for fluid communicationbetween the first cavity and second cavity.

In another embodiment, a method of microfluidic handling is provided.The method includes filling a first cavity defined by a first layer withfluid through a channel; filling a second cavity defined by a secondlayer with fluid through a channel; providing a fluid separation betweenthe first cavity and the second cavity; compressing the first and secondlayers relative to one another to cause sealing of the channelsresulting in obstruction of the first cavity and the second cavity fromfurther fluid entry; and removing the fluid separation and allowing forfluid communication between the first cavity and second cavity.

In yet another embodiment, a microfluidic device is provided. The deviceincludes a first layer defining a first cavity; a first channel arrangedto provide fluid entry to the first cavity; a second layer defining asecond cavity; a second channel arranged to provide fluid entry to thesecond cavity; and at least one separating material providing fluidseparation between the first cavity and the second cavity, wherein thefirst and second channels are adapted to be sealed resulting inobstruction of the first cavity and the second cavity from further fluidentry, and wherein heating or dissolving of the at least one separatingmaterial causes removal of the fluid separation allowing for fluidcommunication between the first cavity and second cavity.

In another embodiment, a method of microfluidic handling is provided.The method includes filling a first cavity defined by a first layer withfluid through a channel; filling a second cavity defined by a secondlayer with fluid through a channel; providing at least one separatingmaterial as a fluid separation between the first cavity and the secondcavity; sealing the channels of the first and second layers resulting inobstruction of the first cavity and the second cavity from further fluidentry; and heating or dissolving the at least one separating material toremove the fluid separation and allowing for fluid communication betweenthe first cavity and second cavity.

In yet another embodiment, a microfluidic device is provided. The deviceincludes a first layer defining a plurality of cavities; a plurality ofchannels arranged to provide fluid entry to the plurality of cavities; asecond layer disposed adjacent to the first layer; and whereincompression of the first and second layers relative to one anothercauses sealing of the plurality of channels resulting in obstruction ofthe plurality of cavities from further fluid entry.

In another embodiment, a microfluidic device is provided. Themicrofluidic device includes a first layer defining at least onechannel; a second layer having a plurality of protrusions disposedadjacent to the at least one channel; and a sealing material disposedadjacent to the at least one channel, wherein the plurality ofprotrusions are adapted to force at least a portion of the sealingmaterial into the at least one channel upon application of pressuretoward the at least one channel, causing partitioning of the at leastone channel into a plurality of separate cavities.

In another embodiment, a method of microfluidic handling is provided.The method includes supplying a fluid to at least one channel defined bya first layer, the at least one channel disposed adjacent to a sealingmaterial and a second layer having a plurality of protrusions; pressingthe plurality of protrusions toward the at least one channel; andforcing at least a portion of the sealing material into the at least onechannel to partition the at least one channel into a plurality ofseparate cavities containing the fluid.

Advantages, novel features, and objects of the invention will becomeapparent from the following detailed description of the invention whenconsidered in conjunction with the accompanying drawings, which areschematic and which are not intended to be drawn to scale. For purposesof clarity, not every component is labeled in every figure, nor is everycomponent of each embodiment of the invention shown where illustrationis not necessary to allow those of ordinary skill in the art tounderstand the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Variousembodiments of the invention will now be described, by way of example,with reference to the accompanying drawings.

FIG. 1 shows a top view of a microfluidic device in accordance with someembodiments;

FIG. 2 illustrates an exploded perspective view of the microfluidicdevice of FIG. 1;

FIGS. 3A-3C depict different layers of the microfluidic device of FIG.1;

FIG. 4 shows a cross-sectional view of the microfluidic device of FIG.1;

FIG. 5 depicts a top view of a microfluidic device in use in accordancewith some embodiments;

FIG. 6 shows a top view of a microfluidic device having one layer filledin accordance with some embodiments;

FIG. 7A shows a top view of a microfluidic device having two layersfilled in accordance with some embodiments;

FIG. 7B illustrates a cross-sectional view of the microfluidic device ofFIG. 7A;

FIG. 8A depicts a top view of a microfluidic device with isolatedcavities in accordance with some embodiments;

FIG. 8B illustrates a cross-sectional view of the microfluidic device ofFIG. 8A;

FIG. 9A shows a top view indicating a cross-section of a microfluidicdevice;

FIG. 9B illustrates a series of cross-sectional views of a microfluidicdevice where the channels are being sealed in accordance with someembodiments;

FIG. 9C illustrates a series of cross-sectional views of anothermicrofluidic device where the channels are being sealed in accordancewith some embodiments;

FIG. 9D depicts a series of cross-sectional views of yet anothermicrofluidic device where the channels are being sealed in accordancewith some embodiments;

FIG. 10A shows a top view of a microfluidic device with combinedcavities in accordance with some embodiments;

FIG. 10B illustrates a cross-sectional view of a microfluidic devicewith a thinned separating material in accordance with some embodiments;

FIG. 10C depicts a cross-sectional view of a microfluidic device withcorresponding cavities combined in accordance with some embodiments;

FIG. 10D depicts another cross-sectional view of a microfluidic devicewith corresponding cavities combined in accordance with someembodiments;

FIG. 10E shows another cross-sectional view of a microfluidic devicewith corresponding cavities combined in accordance with someembodiments;

FIG. 10F illustrates another cross-sectional view of a microfluidicdevice with where fluid ingredients in corresponding cavities are mixedin accordance with some embodiments;

FIG. 11A shows an exploded perspective view of portions of yet anothermicrofluidic device in accordance with some embodiments;

FIG. 11B illustrates a cross-sectional view of the microfluidic deviceof FIG. 11A;

FIG. 12A shows an exploded perspective view of portions of anothermicrofluidic device in accordance with some embodiments;

FIG. 12B illustrates a cross-sectional view of the microfluidic deviceof FIG. 12A;

FIG. 13A shows a top view close-up of a microfluidic device inaccordance with some embodiments;

FIG. 13B depicts a perspective view close-up of a microfluidic device inaccordance with some embodiments;

FIG. 14 depicts a perspective view close-up of a microfluidic device inaccordance with some embodiments;

FIG. 15 illustrates a microfluidic device coupled with a thermal cyclingassembly in accordance with some embodiments;

FIG. 16A depicts an exploded perspective view of another microfluidicdevice in accordance with some embodiments;

FIG. 16B shows a cross-sectional view of the microfluidic device of FIG.16A;

FIG. 16C shows a cross-sectional view of the microfluidic device of FIG.16A with sealed channels;

FIG. 17A depicts an exploded perspective view of yet anothermicrofluidic device in accordance with some embodiments;

FIG. 17B shows a cross-sectional view of the microfluidic device of FIG.17A;

FIG. 17C shows a cross-sectional view of the microfluidic device of FIG.17A with sealed channels;

FIG. 18A depicts an exploded perspective view of another microfluidicdevice in accordance with some embodiments;

FIG. 18B shows a cross-sectional view of the assembled microfluidicdevice of FIG. 18A;

FIG. 19A depicts an exploded perspective view of another microfluidicdevice in accordance with some embodiments;

FIG. 19B shows a cross-sectional view of the assembled microfluidicdevice of FIG. 19A;

FIG. 20A depicts an exploded perspective view of another microfluidicdevice in accordance with some embodiments;

FIG. 20B shows a cross-sectional view of the assembled microfluidicdevice of FIG. 20A;

FIG. 21 depicts an exploded perspective view of another microfluidicdevice in accordance with some embodiments;

FIG. 22A depicts an exploded perspective view of another microfluidicdevice in accordance with some embodiments;

FIG. 22B shows a cross-sectional view of the assembled microfluidicdevice of FIG. 22A;

FIG. 22C shows a cross-sectional view of the assembled microfluidicdevice of FIG. 22B in a different state; and

FIG. 23 shows a cross-sectional view of another microfluidic device inaccordance with some embodiments.

DETAILED DESCRIPTION

The present disclosure relates to microfluidic devices that provide formultiple combinations to be made of a number of groups of fluidingredients, inexpensively and efficiently. For instance, in alaboratory setting, a user faced with multiple groups of reagents thatare to be combined in every possible permutation for combinatorialanalysis (e.g., PCR detection) may easily implement embodiments ofmicrofluidic devices discussed herein to achieve this goal.

The present disclosure also relates to microfluidic devices that allowfor a fluid ingredient to be divided evenly into separate volumes (e.g.,through partitioned cavities). Those separate volumes may be fluidlyisolated from one another in a quick and easy manner.

In some embodiments, when in use, a microfluidic device may perform acombinatorial analysis between different groups of fluid reagents (e.g.,a group of DNA samples and a group of DNA primers). Such a combinatorialanalysis may be performed via an array of chambers where each chamberincludes at least two cavities that are, initially, fluidly separated.For instance, a plurality of first cavities of one layer may beseparated by a separating material from a plurality of second cavitiesof another layer. The plurality of first cavities may be appropriatelyfilled with fluid from a first group of fluid source reservoirs (e.g.,DNA samples). The plurality of second cavities may also be appropriatelyfilled with fluid from a second group of fluid source reservoirs (e.g.,DNA primers).

Accordingly, the device may facilitate every combination between thedifferent groups of fluid reagents (e.g., DNA samples and DNA primers).Once respective cavities are suitably filled, each cavity of eachchamber is fluidly isolated from one another. Subsequently, the fluidseparation between corresponding cavities of each chamber is removed,allowing fluids located within corresponding cavities to mix. Uponmixing, various conditions leading to appropriate reactions (e.g., PCR,biological reactions, chemical reactions, etc.) may be implemented.

Microfluidic devices of the present disclosure may include a number oflayers where each layer defines a number of cavities. A number ofchannels may be arranged to provide fluid entry to the cavities. In someembodiments, certain layers may define both the cavities and thechannels. In some embodiments, separate layers may define separatecavities and channels. Further, in some cases, a separating material mayprovide fluid separation between a number of the cavities, such ascavities that correspond to a reaction chamber.

When the cavities are adequately supplied with appropriate fluidingredient(s), the channels that initially provide fluid connectionbetween the cavities may be sealed so as to prevent further fluid entryinto the cavities. In some embodiments, the layers of the device may becompressed relative to one another (e.g., due to external clamping,probing or rolling of the device) so as to cause the channels to seal.For example, such compression may cause a sealing material to bedeformed into the space defined by the channel.

In situations where a separating material divides corresponding cavitiesof a reaction chamber and it is desired for the contents within thosecavities to mix, the separating material may be suitably manipulated sothat the fluid separation between cavities is removed. Such removalallows for mixing of the respective fluid ingredients of thecorresponding cavities. In some embodiments, portions of the separatingmaterial may be heated or dissolved so that the fluid separation betweencorresponding cavities of a chamber is removed.

Microfluidic devices described herein may include an array of reactionchambers. Initially, each chamber may include one or more fluidseparation(s) (e.g., a separating material such as a film, a membrane,etc.) between a number of cavities. In some embodiments, a separatingmaterial is disposed between layers that define cavities and/orchannels, allowing for fluid enclosure within the cavities and channels.The separating material may further divide reaction chambers of thedevice into corresponding cavities, preventing fluid from otherwiseflowing between the corresponding cavities of each chamber. Thus, whilethe cavities of each chamber may be individually filled with a fluidingredient, the fluid ingredients located within each of thecorresponding cavities of the chamber are unable to be mixed as long asthe fluid separation is present to divide the cavities.

Before removal of the fluid separation between corresponding cavities ofthe same chamber, if removal occurs at all, neighboring cavities (e.g.,cavities defined by an appropriate layer of the device) may be fluidlyisolated (e.g., sealed or obstructed from further fluid entry) from oneanother. Such fluid isolation may occur through deformation (e.g.,plastic deformation) of a suitable sealing material into channels thatwould otherwise provide fluid connection between the cavities.

In some embodiments, the separating material itself may be the sealingmaterial that is deformed so as to cause each of the cavities of thedevice to be fluidly isolated from one another. For instance,appropriate portions of the separating material, which also act as asealing material, may deform into channels causing a seal to obstructfluid from flowing between neighboring cavities connected by thechannels. The separating material may be further deformed so as tocreate an opening between corresponding cavities of each reactionchamber. Once an opening is created between corresponding cavities of achamber, the chamber is no longer under fluid separation and, as aresult, the contents located within each of the corresponding cavitiescan be combined.

Aspects of the present disclosure provide for arrangement(s) and/ortechnique(s) for making the process of combining different groups ofreagents into separate reaction chambers quick and efficient. Aspectspresented herein also provide for even division or partitioning of afluid ingredient into small volumes and along with fluid isolation ofthose small volumes. It can be appreciated that microfluidic devicesdescribed herein may include any suitable number of layers, components,separating materials, sealing materials, adhesives, etc., in anyappropriate arrangement or configuration, and that particular examplesof the present disclosure are not intended to be limiting.

FIG. 1 depicts an embodiment of a microfluidic device 10 having an arrayof chambers 12 where each chamber is divided into corresponding cavities102, 112 (separate cavities 102, 112 are further illustrated in FIGS. 3Aand 3C). Inlets 20, 30 and vents 22, 32 provide for appropriate fillingof fluid ingredients into respective groups of cavities, as described inmore detail further below, for subsequent reaction.

FIG. 2 depicts an exploded view of this embodiment showing themicrofluidic device 10 to include a first layer 100, a second layer 110and an intermediate layer 150 disposed in between the first and secondlayers. The intermediate layer 150 acts as a separating material thatprovides fluid separation between corresponding cavities that make upeach chamber. As discussed further below, the intermediate layer 150 canalso act as a sealing material for fluidly isolating each of thecavities from one another.

FIG. 3A depicts the first layer 100 which defines a number of cavities102 that are divided into subsets of cavities. Each subset of cavitiesincludes a respective inlet 20, for supply of a fluid ingredient to eachof the cavities, and vent 22, for exhaust of excess fluid (e.g., air,ingredient(s), etc.) when appropriate. Illustrated in FIG. 3A, tendifferent fluid source reservoirs are provided as respective inlets forfilling each of the subsets of cavities. That is, the first layer hasten inlets 20 a, 20 b, 20 c, 20 d, 20 e, 20 f, 20 g, 20 h, 20 i, 20 jwhere each inlet is in fluid connection with five cavities 102 and vents22. Respective inlets 20, cavities 102 and vents of a subset are, inturn, connected to one another via a plurality of channels 104.

For example, a fluid ingredient may be supplied through inlet 20 a toeach cavity of the subset of cavities 102 a ₁, 102 a ₂, 102 a ₃, 102 a₄, 102 a ₅ via a channel 104, which allows the cavities 102 a ₁, 102 a₂, 102 a ₃, 102 a ₄, 102 a ₅ to be in fluid communication duringfilling. Accordingly, different fluid ingredients may be supplied toeach subset of cavities corresponding to the inlets 20 a, 20 b, 20 c, 20d, 20 e, 20 f, 20 g, 20 h, 20 i, 20 j. Inlets 30 and corresponding vents32 are also provided in first layer 100 so as to reach through to thesecond layer 110 for providing suitable fluid lines to supply cavitieswithin the second layer.

As provided herein, channels 104 may include any part of the passagewaythrough which fluid may flow, for example, between an inlet 20 and avent 22, or between two or more of the cavities 102. Thus, a singlechannel may be considered to include multiple cavities by virtue of thecavities being in fluid communication. For example, a channel 104 mayinclude each of cavities 102 a ₁, 102 a ₂, 102 a ₃, 102 a ₄, 102 a ₅which are in fluid communication.

FIG. 3B shows an intermediate layer 150 that includes a separatingmaterial having holes that form part of the inlets 30 and vents 32leading to second layer 110. That is, the holes of the intermediatelayer 150 allow for fluid connection between respective fluid sourcereservoirs that are provided as inlets 30 originating at the first layer100 and respective cavities 112 and channels 114 of the second layer.Accordingly, in this embodiment, fluid ingredient may be suppliedthrough the first layer 100 to fill appropriate cavities 112 andchannels 114 of the second layer 110. Further, the vents 32 may allowexcess fluid from the second layer to escape through the first layer.

FIG. 3C depicts the second layer 110 which, similar to the first layer100, defines a number of cavities 112 divided into subsets of cavities.Each subset of cavities includes a respective inlet 30 and vent 32 sothat fluid ingredients may be appropriately supplied to each of thecavities. As shown in FIG. 3C, the second layer has five inlets 30 a, 30b, 30 c, 30 d, 30 e where each inlet is in fluid communication with tencavities 112 and vents 32 which are each, in turn, connected to oneanother via respective channels 114.

For instance, a fluid ingredient may be supplied through inlet 30 a toeach cavity of a subset of cavities 112 a ₁, 112 a ₂, 112 a ₃, 112 a ₄,112 a ₅, 112 a ₆, 112 a ₇, 112 a ₈, 112 a ₉, 112 a ₁₀ via channel 114.During filling, the channel 114 provides fluid connection between eachof the cavities 112 a ₁, 112 a ₂, 112 a ₃, 112 a ₄, 112 a ₅, 112 a ₆,112 a ₇, 112 a ₈, 112 a ₉, 112 a ₁₀. It follows that different fluidingredients may be supplied to each subset of cavities corresponding tothe inlets 30 a, 30 b, 30 c, 30 d, 30 e.

Similarly to that described above with respect to channels 104, channels114 may include any part of the passageway through which fluid may flow,for example, between an inlet 30 and a vent 32, or between two or moreof the cavities 112. For instance, a channel 114 may include each ofcavities 112 a ₁, 112 a ₂, 112 a ₃, 112 a ₄, 112 a ₅, 112 a ₆, 112 a ₇,112 a ₈, 112 a ₉, 112 a ₁₀ which are in fluid communication.

It can be appreciated that the above embodiment is presented by way ofexample only and is not meant to limit the present disclosure. Forexample, microfluidic devices described herein may include any suitableconfiguration of layers and components that provide for an appropriatearrangement of cavities and/or channels that may facilitate separatereactions in the cavities, or chambers that include one or morecavities.

In some embodiments, certain regions of the intermediate layer 150 orthe entire layer itself accommodate adherence or attachment of the firstand second layers together. For example, the layer may include one ormore materials (e.g., adhesive, adhesive on a backing, etc.) thatfacilitate bonding between the layers. In some embodiments, a differentlayer and/or material not explicitly shown in the figures is used toadhere various layers together.

In some embodiments, portions of the intermediate layer 150 may form abarrier on one side (e.g., the bottom) of the first layer 100 so as toappropriately enclose the cavities 102 and channels 104. Conversely,portions of the intermediate layer 150 may form a barrier on one side(e.g., the top) of the second layer 110 so as to provide suitableenclosure for the cavities 112 and channels 114. Accordingly, theintermediate layer 150 (e.g., separating material) may provide fluidseparation of a chamber into corresponding cavities (e.g., a firstcavity from the first layer and a second cavity from the second layerthat are in alignment with one another).

The cross-sectional view depicted in FIG. 4 shows an embodimentincluding a number of chambers with corresponding cavities of eachchamber.

Channels connect cavities associated with respective layers together. Asshown, a first layer 100 and a second layer 110 are respectively bondedto the intermediate layer 150 on either side. The intermediate layer 150provides for a separating material that forms a fluid separation betweencorresponding cavities of each chamber. In some embodiments, the firstand second layers are generally stiffer (e.g., relatively more rigid)than the intermediate layer. In other embodiments, the first and secondlayers are of a similar stiffness, or less stiff, than the intermediatelayer. The first and second layers, as compared with one another, may beof a similar or different stiffness.

As shown in FIG. 4 and indicated by the dashed circle, the chamber 101includes a first cavity 102 of the first layer and a second cavity 112of the second layer, divided by the separating material of theintermediate layer. The channel 104 provides a fluid connection betweenother cavities 102 of the first layer. Similarly, the channel 114 (whichis not expressly shown as it comes through the cross-sectional plane)provides a fluid connection between other cavities 112 of the secondlayer.

While the intermediate layer 150 is depicted as a single monolithicmembrane that serves as a separating material between correspondingcavities of each chamber as well as forming a barrier to form a suitableenclosure for respective cavities 102, 112 and channels 104, 114, it canbe appreciated that the intermediate layer may include a number ofcomponents that are separate from one another. In some embodiments, theintermediate layer may include individual separating material componentsthat are placed at discrete positions relative to respective cavitiesand channels of the first and second layers. For example, theintermediate layer may include a particular separating material, orportions thereof, that are appropriately positioned so as to define thecavities, and a different separating material, or portions thereof, thatdefine the channels. Such an arrangement may be beneficial when it ispreferred that various regions of the separating material exhibitdifferent properties (e.g., more rigid, more soft) depending on whetherthe material is located directly adjacent to a cavity, or a channel.

A separating material may be composed of any suitable material orcomposition. In some embodiments, the separating material includes aplastic/polymer, such as polyethylene, polypropylene, ethylene-vinylacetate, an elastomer such as silicone rubber, or a wax such as paraffinor microcrystalline wax, or combinations thereof. As such, theseparating material may include a blend of several differentcompositions or may be provided in various configurations, such as inlayered or more discrete arrangements, where different pieces of theseparating material are placed at various locations. As discussedfurther below, during use, the separating material may also be used as asealing material for preventing fluid flow between cavities throughconnecting channels. Or, a different sealing material may be used incombination with a distinct separating material.

Layers of microfluidic devices described herein may include any suitablematerial and may be composed of similar or different materials. In someembodiments, a layer is made of a relatively rigid plastic, for example,polypropylene, polyethylene, polycarbonate, PTFE, etc. Alternatively, alayer may include a flexible, rubber-like material such as silicone orother elastomer. Other potential materials include glass, ceramics,silicon, or the like. As such, a layer may be rigid or deformable. Suchmaterials may be translucent or clear so as to easily allow for opticalmeasurements of the contents within the chambers/cavities.

Individual layers of the microfluidic device may be made by any suitablemethod. In some embodiments, layers are fabricated via injectionmolding, by embossing the cavities and channels into a thin sheet ofplastic, etching, or any other suitable method. For example, spaces thatdefine the cavities and channels may be formed (e.g., molded, etched) ina plastic/polymer or elastomeric material that makes up the layer.

In some embodiments, different layers may define cavities and channelsthat are initially in fluid communication. For example, a first layermay define a number of cavities without defining the channels thatconnect the cavities together; an additional layer adjacent to the firstlayer may define those channels that connect the cavities. Such channelsmay be appropriately sealed, for example, by compression of the twolayers relative to each other.

As such, a layer may define one or more cavities and/or channels of thedevice, such as in embodiments that depict the first and second layers100, 110. Or, as also discussed above, a layer may include a separatingmaterial and/or a sealing material, such as the intermediate layer 150.

Accordingly, in some embodiments, a layer may include acrylic adhesive,natural rubber adhesive, or silicone adhesive. Such materials may besuitable to deform into channels of the device (e.g., as a sealingmaterial) when subject to compression. In some embodiments, adhesivesmay be disposed on a relatively rigid layer, or alternatively, on aseparate backing Examples of suitable backings may includepolypropylene, polyethylene, polycarbonate, and/or other suitableplastics.

Various components (e.g., layers, adhesives, etc.) of microfluidicdevices described herein may be adhered together by any suitable method.For example, an adhesive may be used to bond one or more componentstogether, such as for bonding a separating/sealing material and thefirst and/or second layers. In some embodiments, the components of themicrofluidic device are compressed together (e.g., via clamping,rolling, or other externally applied force) so as to result in an evenlydistributed bond between surfaces of different layers.

For certain materials, the application of an appropriate amount ofcompression and/or heat may result in certain characteristics of theseparating/sealing material, or other components of the device, tochange. For example, at elevated temperatures, certain materials such aswax will become increasingly tacky and/or adhesive, resulting in strongadherence between components of the device. Accordingly, the differentlayers of the device, including a wax layer, may be assembled and thensubject to compression and heating for an appropriate period of time,allowing the wax to create a bond. In some embodiments, one or moreappropriate solvents may be used to promote bonding between layers.

As discussed above, microfluidic devices according to the presentdisclosure may be useful to facilitate multiple combinations ofdifferent groups of reagents. In some embodiments, once fluidingredients of corresponding cavities in various chambers arecombined/mixed, the chamber(s) may be subject to thermal cycling. Forexample, a heat source configured for thermal cycling (e.g., tofacilitate PCR) may be placed in contact, or otherwise coupled, with thefirst and/or second layer. In some cases, the layer in which the heatsource is in contact may be relatively thin and/or may include athermally conductive material so as to facilitate heat transfer.

Referring to the embodiment of FIGS. 1-4, the first layer 100 providesten inlets 20 with five cavities 102 fluidly connected to eachrespective inlet 20 and the second layer 110 provides five inlets 30with ten cavities 112 fluidly connected to each respective inlet 30.Accordingly, the device may accommodate a total of fifty differentreactions based on mixtures of different combinations of reagents,depending on the supply of various fluid ingredients. Takingcombinatorial PCR reactions described previously as an example, each ofthe inlets 20 of the first layer 100 may be supplied with ten differentDNA samples to be analyzed and, hence, each of the inlets 30 of thesecond layer 110 may be supplied with five different DNA primer mixes astarget probes for analysis of the respective DNA samples.

FIGS. 5-10F illustrate embodiments of the present disclosure where eachof the cavities of the device are supplied with respective fluidingredients prior to fluid isolation of the cavities, and after fluidisolation of the cavities, subsequent mixing of the contents withincorresponding cavities.

FIG. 5 shows an embodiment of an assembled microfluidic device where anumber of fluid source reservoirs are provided as inlets 20, 30. A groupof fluid source reservoirs 20 are arranged to provide filling ofrespective cavities of the first layer 100 with fluid ingredient 500.And another group of fluid source reservoirs 30 are arranged to fillrespective cavities of the second layer 100 with fluid ingredient 502.By way of explanation, fluid ingredients 500 a, 500 b, 500 c, 500 d, 500e, 500 f, 500 g, 500 h, 500 i, 500 j filling inlets 20 of the firstlayer 100 are illustrated to be lighter in color than fluid ingredients502 a, 502 b, 502 c, 502 d, 502 e filling inlets 30 of the second layer110. FIG. 6 illustrates cavities of the second layer 110 having beenfilled with fluid ingredients 502 a, 502 b, 502 c, 502 d, 502 e fromrespective fluid source reservoirs.

FIG. 7A shows cavities of the first layer 100 having been filled withfluid ingredients 500 a, 500 b, 500 c, 500 d, 500 e, 500 f, 500 g, 500h, 500 i, 500 j from respective fluid source reservoirs. The fluidsource reservoirs are provided as inlets 20 and arranged to fillrespective cavities 102.

Fluid ingredients may be supplied to respective inlets, cavities andchannels by any suitable method. In some embodiments, a machine or userpipettes appropriate reagents into input wells. Or, separate containersmay be appropriately connected to respective inlets for supplying fluidheld by the containers to the inlets. Cavities and channels may besupplied with respective fluid ingredients, for example, by capillaryaction, gravity, centrifuging, suction from the vent holes, vacuumsuction, pressure on the input wells, and/or any other suitable manner.Thus, any push or pull method may be used to suitably supply cavitiesand channels with appropriate fluid ingredient(s). Fluid ingredients 500and 502 may be supplied to cavities 102 and 112 sequentially orsimultaneously.

Each input well 20 of the first layer 100 of this example may besuitable to accommodate filling of five cavities and the connectingchannels between those cavities. In some cases, an amount of extra fluidingredient may be left over. Similarly, each input well 30 of the secondlayer 110 may be suitable to accommodate filling of ten cavities and theconnecting channels of those cavities. Vent holes may allow for excessair and/or fluid ingredient to escape from the channels and/or cavitieswhen the channels and cavities are filled with respective fluidingredients and be exhausted from the system. For instance, compressionof the first and second layers relative to one another may reduce theamount of space available and/or increase the fluid pressure within thedevice (e.g., within cavities and channels) resulting in fluiddisplacement; hence, vent holes may accommodate for an appropriateamount of overflow to occur.

The cross-sectional view depicted in FIG. 7B shows correspondingcavities of each chamber filled with fluid ingredients 500, 502. Asindicated by the dashed circle, the chamber 101 includes first cavity102 and second cavity 112 which are, at this stage, divided by theseparating material of the intermediate layer 150 such that fluid isunable to flow between the cavities, hence, mixing of the fluidingredients 500, 502 with each other is prevented. As further shown inFIG. 7B, the channel 104 provides a fluid connection between othercavities of the first layer having received fluid ingredient from arespective inlet 20. Similarly, the channel 114 (not expressly shown)provides a fluid connection between other cavities 112 of the secondlayer having received fluid ingredient from a respective inlet 30.

After the cavities are filled with the appropriate fluid ingredients,the cavities are subsequently isolated from one another such that fluidis unable to spill or flow from one cavity to another. In bringing aboutthis fluid isolation, in some embodiments, a sealing material is causedto deform into and substantially fill the space defined by the channelsso that fluid flow that was previously permitted between cavities is nowblocked.

To cause such sealing of the channels between cavities, in someembodiments, an external force (e.g., compressive) and/or heat isapplied to the device. For example, the device (e.g., at exteriorsurfaces of the layers) may be clamped, rolled or probed so as to besubject to a suitable compressive force. Further, the device may beheated so as to cause deformation of the sealing material itself, which,in some embodiments, also happens to be the separating material.

FIGS. 8A-8B show an embodiment where application of an appropriatedegree of compressive force and/or heat pushes certain portions of theseparating/sealing material (e.g., regions of the separating/sealingmaterial aligned with the channels) into the channels, resulting insealing of the channels and, thus, fluid isolation of the cavities fromone another.

In some embodiments, the amount of compressive force exerted on thedevice sufficient to result in sealing off of the channels may bebetween about 100 Newtons and about 10,000 Newtons (e.g., 100-1,000 N;1,000-5,000 N; 5,000-10,000 N), depending on the overall size of thedevice and the nature of the separating/sealing material. For example,the larger the device (i.e., the more cavities and channels), the morecompressive force may be required to seal the channels from flow betweencavities. Or, the softer the material, the less compressive force may beneeded to deform the material into respective channels of the device.

In some embodiments, the separating/sealing material may be heated to anelevated temperature so as to cause softening of the material sufficientfor the material to deform into adjacent channels. Such an elevatedtemperature may depend on the softening point of the material, forexample, between about 40 C and about 90 C (e.g., between about 50 C andabout 70 C) for certain polymeric or wax compositions.

It can be appreciated that any suitable method may be used to create aseal within channels such that cavities are fluidly isolated from oneanother. For example, separate pieces of a sealing material may bedistributed throughout the device and located directly adjacent to eachchannel. Upon application of heat or laser irradiation, locally (e.g.,with a heat probe) and/or globally (e.g., with a heated plate) to theoverall device, the sealing material may melt, soften, or otherwisedeform into the channel so as to cause sealing of the channel resultingin obstruction of fluid flow between cavities. For example, the sealingmaterial may be adapted to, at least partially, melt, soften, orotherwise deform into the channel upon application of externalcompressive force and/or heat to particular regions of the device, orthe device as a whole.

The cross-sectional view provided by FIGS. 9A-9B show a process ofsealing a channel 114 upon appropriate actuation (e.g., compression,heating and/or other suitable method) of the sealing material. FIG. 9Ashows where the cross-section is taken to illustrate FIG. 9B. In FIG.9B, the intermediate layer 150 disposed between first and second layers100, 110 includes a separating/sealing material.

The first and second layers 100, 110 are compressed relative to oneanother such that a portion 151 of the separating/sealing material iscaused to deform into the space defined by the channel through whichfluid would otherwise be able to flow. As shown, other portions of theseparating/sealing material are also compressively deformed as well,depicted by the reduction in thickness t of the intermediate layer 150.In some embodiments, some of the portion 151 of the separating/sealingmaterial that deforms into the channel 114 is supplied byseparating/sealing material displaced from areas surrounding thechannels, which contributes to the overall reduction in thickness t ofthe intermediate layer 150. The portion of the separating material 151that blocks the channel, however, prevents fluid from flowing throughthe channel 114 between cavities that were previously in fluidcommunication.

In another embodiment, a portion of the first or second layer(s) itselfmay be deformed into the channel so as to cause sealing. For instance,the first or second layer may be composed of a deformable material, suchas silicone, an elastomer, or the like. Such a material may act as asealing material. Or, the first and/or second layer may include amaterial that substantially softens after the application of heat sothat the material deforms into the channels.

In some embodiments, adjacent layers are compressed relative to oneanother and material from one of the layers itself behaves as a sealingmaterial and deforms into the channel space. As shown in FIG. 9C,appropriate actuation of the first or second layer(s) may cause channelcollapse, that is, deformation of a portion of the sealing material 116into the space that previously permitted fluid flow there through. Suchcollapse prevents fluid from flowing through the channel 114 betweencavities. Accordingly, fluid located within neighboring cavities thathad previously been in fluid communication is no longer able to flowbetween the cavities.

In another embodiment, the intermediate layer 150 may include a distinctseparating material 152 and a sealing material 154 for sealing thechannels 104, 114. FIG. 9D depicts an example where the sealing material154 is disposed directly adjacent to the channels between the separatingmaterial 152 and the channel 114.

Appropriate actuation of the sealing material 154 (e.g., application ofcompressive force, heating, magnetic force, etc.) may cause a portion ofthe sealing material 154 to be deformed into the channel 114 such thatfluid is unable to flow through the channel 114 and between cavities. Insome cases, such a sealing material 154 may be more susceptible todeformation (e.g., may have a lower compressive and/or thermal thresholdfor softening/deforming) than the separating material 152. For instance,when the layers are subject to compression relative to one another(e.g., due to an externally applied force), for this embodiment, theapplied compressive force causes the portion of the sealing material 156to deform into the channel while the separating material 152 of theintermediate layer 150 remains largely without any deformation, if atall.

In some embodiments, the process of sealing the channels isirreversible. For example, a sealing material may be plastically, hence,permanently deformed into the space of the channel through which fluidwould otherwise be able to flow. Accordingly, absent disassembly of themicrofluidic device to remove the sealing material from the channels,such channel sealing is not a reversible characteristic.

It can be appreciated that alternative methods of actuation may cause asealing material to deform into the channel, sealing off fluid flowbetween cavities.

While separate and distinct compositions may be used as a separatingmaterial and a sealing material, as described above, the samecomposition may be used as both a separating material and a sealingmaterial. For example, an intermediate layer 150 may include a membranecomposed of a suitable composition (e.g., polymer, wax, or combinationsthereof) that may serve to cause sealing of channels between cavities(e.g., by deformation into the channels) as well as to separatecorresponding cavities of a reaction chamber from one another prior tomixing. Or, as also discussed with reference to FIG. 9C, a layer thatdefines cavities and/or channels may itself include a composition thatserves as a sealing material to prevent fluid flow between neighboringcavities.

In some cases, any portion of the intermediate layer 150, such as theseparating material 152 and/or the sealing material 154, may be used asan adhesive for bonding layers of the device together.

For instances where channels are sealed due to deformation of a sealingmaterial into neighboring channels, any suitable methods for causing thedeformation may be used. As discussed above, a sealing material may becaused to appropriately deform due to compressing layers of the device(e.g., externally applied compression, clamping/rolling of exteriorsurfaces of the layer(s), applying a local probe to various regions,etc.), heating appropriate portions of the device (e.g., using a thermalplate, a heat probe, a laser, etc.), magnetically actuating certainregions of the device, or any other suitable method. It can beappreciated that different regions of the device may be selectivelyactuated. For example, heat and/or compression may be directed toparticular regions where sealing of the channels is desired, rather thanto an entire surface of the device.

Once the cavities are sealed from one another, the separating materialdividing corresponding cavities of each chamber may be appropriatelymanipulated so that the fluid ingredients 500, 502 within the cavitiesthat make up a chamber are permitted to be combined. FIG. 10A shows anembodiment where openings are created in the separating materialresulting in a mixture 504 of fluid ingredients of correspondingcavities of each chamber (shown by the shading of the chambers).

In some embodiments, the strength of the separating material may besubstantially reduced when subject to a certain temperature range (e.g.,above room temperature), for example, between about 20 C and about 100C, or between about 50 C and about 90 C. Thus, when the device, orparticular sections of the separating material, is heated to an elevatedtemperature, the portion of the separating material that divides eachchamber may be prone to tearing (e.g., tearing upon application of arelatively small amount of stress), allowing contents withincorresponding cavities of each chamber to mix.

In some embodiments, the separating material may be suitably weakenedduring manufacture/assembly of the microfluidic device. For example, theseparating material may be pre-stressed (e.g., as would be the case witha shrink film) or thinned prior to assembly of various parts of themicrofluidic device. Or, a separating material that includes a polymer(e.g., polyethylene, polyester, polypropylene) and/or paraffin wax maybe appropriately stretched prior to attachment of the differentlayers/layers of the microfluidic device. In some embodiments, theseparating material is attached to different adjacent layers while it isin its stretched state, so that the material is under stress in theassembly. Alternatively, certain features that serve to weaken theseparating material (e.g., scoring/cut/perforated configurations) whileretaining fluid separation between cavities may be added at particularlocations (e.g., regions that divide each chamber into correspondingcavities).

In some embodiments, by virtue of different layers of the device beingcompressed during sealing of the channels, the separating material maybe weakened at the edge of each chamber. For instance, in FIG. 10B, theintermediate layer 150 includes a separating/sealing material thatserves to seal the channels between cavities and also to provide initialfluid separation between corresponding cavities of a reaction chamber.When the channels between cavities are sealed, the separating materialis depicted in FIG. 10B to be relatively thinner in regions surroundingthe cavities, for example, due to compression of the layers relative toone another, heating and/or any other method. That is, as portions ofthe separating material are compressed during sealing of the channels,the separating material may be made relatively thinner at the edges ofchambers as compared to in the middle of the chamber where nocompressive force is applied. Accordingly, the separating material inthe center of the chamber will remain at its original thickness.

As a result, the separating material may be more prone to tearing atedges of the cavities. Thus, when the separating material is heated,dissolved, compressed and/or otherwise suitably manipulated, openingsmay be formed in the separating material (e.g., from tearing) at thethinner/weakened sections near the edges of chambers. Such openingsallow fluid ingredients 500, 502 to combine forming a mixture 504, asfurther shown in FIGS. 10C-10F. In some embodiments, a torn portion ofthe separating material 156 remains within the chamber.

In some embodiments, to facilitate tearing, as discussed above, theseparating material may be stretched prior to adherence to the first andsecond layers. Placing the separating material under a pre-stress mayallow for subsequent manipulation of the material to stretch, tearand/or deform in a controlled manner. For some materials, such as shrinkfilm, the material may be stretched during manufacturing. For example,upon the application of heat, the material shrinks. Such shrinking maypromote tearing of the material and may also serve to widen openings inthe material, facilitating further fluid communication between the firstand second cavity. Alternatively, if the separating material is in astretched state while being attached to different adjacent layers,larger openings may be created than would otherwise form uponappropriate manipulation (e.g., heating, dissolving). Such stress mayfurther promote tearing of the separating material.

When appropriate, the separating material may be pre-stretched atregions apart from certain channels of the device, or pre-stretchedapart from as many channels as possible, so as to decrease thepossibility of undesirable tears forming along or over channels thatwould break the seal between cavities. In some embodiments, thedirection of stretch in the separating material can be chosen so that itdoes not extend substantially parallel to selected channels in order toreduce folding/creasing of the material into these channels.

As discussed previously, the separating material may be a continuoussheet or membrane. Though, the separating material may also include anumber of individual components that are separate and distinct from oneanother. As such, individual components of the separating material mayinclude different material compositions. For example, as discussedpreviously, the portion of a separating material that is located betweencorresponding cavities of a chamber may include a heat sensitivecomposition that is susceptible to deformation (e.g., tearing) uponexposure to elevated temperatures.

Alternatively, the portion of a separating material that is locatedbetween corresponding cavities of a chamber may include a resorbablecomposition (e.g., polyethylene glycol, PLA, PLGA, etc.) that may bedissolved, at least partially, when exposed to an appropriateenvironment (e.g., aqueous, solvent). For example, portions of theseparating material may be soluble in certain solutions such that uponexposure to the solution, the soluble portion dissolves, leaving anopening between previously divided cavities of a reaction chamber. Insome embodiments, the solution that instigates dissolving of theseparating material may be the fluid ingredient(s) supplied to thecavities itself. In some embodiments, the separating material, orportions thereof, may be substantially soluble when exposed toparticular fluids above a certain temperature; thus, heating of thematerial may increase its solubility.

It can be appreciated that a number of different types of separatingmaterials may be used.

In some embodiments, the separating material may include an adhesivecomposition. While regions of the separating material that provide fluidseparation between corresponding cavities of a chamber may be removed,surrounding areas of the separating material may include an adhesive(e.g., silicone adhesive) that assists in bonding layers together. Suchan adhesive may also flow upon compression of the device for sealing ofthe channels.

In some embodiments, the separating material is embedded with aferromagnetic or magnetic material. Accordingly, after filling and fluidisolation of the cavities, the separating material may be placed underan external magnetic field to create a stress that results in thecreation of an opening in the separating material that permits fluidingredients contained within corresponding cavities of a chamber to becombined.

In some embodiments, laser irradiation may be used to form openings inthe separating material. For example, the separating material may beadapted to absorb a greater intensity of radiation at a particularwavelength range (e.g., ultraviolet, infrared, visible) than wavelengthsoutside of this range. In some cases, the material that defines thechannels and cavities (e.g., material of a layer that has the relativespace of cavities and/or channels etched in) may be substantially lessabsorptive of radiation at wavelengths within this range, allowingradiation to pass through the material without substantial heating ordeformation. Such a characteristic allows for a majority of theradiation to be absorbed by the separating material, rather than havingbeen attenuated by a different absorptive material. In some cases,treatment of the separating material with a dye having a high absorptionratio within a particular wavelength range may impart the material withselective absorption properties. Accordingly, when a separating materialtreated with a wavelength-sensitive dye is exposed to laser irradiationat a wavelength range corresponding to the relatively high absorptionfor the dye-treated separating material, openings may form in thoseportions of the separating material exposed to the laser irradiation.

In various embodiments, sealing of channels between cavities occursbefore openings are created between corresponding cavities of eachchamber. That is, after fluidly connected cavities defined by the samelayer are appropriately filled, the cavities are fluidly isolated fromone another as well as other cavities in the same layer. In someembodiments, such fluid isolation of the cavities occurs prior tocombination of fluid within corresponding cavities of a chamber. Thisorder of process may be advantageous because if openings are formed inthe separating material between corresponding cavities of a chamberbefore neighboring cavities are fluidly isolated from one another, thenfluid from those cavities may undesirably leak into surroundingcavities.

In some cases, sealing of channels to fluidly isolate neighboringcavities of the same layer (e.g., via deformation of aseparating/sealing material) and creation of openings (e.g., tearing) ofseparating material between corresponding cavities from different layersmay happen simultaneously, or fluid isolation may occur soon aftercombining corresponding cavities of the chamber. Though, to mitigatecross-contamination of fluid ingredients, sealing of channels betweencavities of the same layer would happen before respective fluidingredients are able to diffuse or mix undesirably into neighboringcavities.

Referring back to the example of FIG. 4, once openings are formed in theseparating material of each chamber so as to allow mixing of thecontents of the two corresponding cavities, fifty separate combinationsof the reagents supplied to respective inlets 20, 30 are created. For aseries of PCR reactions, once appropriate mixing has occurred, thedevice is then ready for thermal cycling. In this example, only threelayers (i.e., first layer 100, second layer 110 and intermediate layer150) are used to produce fifty combinations of reagents (any appropriateset of combinations of reagents may be made) where each chamber is alsoin fluid isolation from the other chambers.

With such a simple construction, a disposable microfluidic device can beeasily manufactured, quickly and at low cost. In some embodiments, oneor more of the layers of the microfluidic device is disposable; or oneor more layers may be reusable after suitable washing to remove residualreagents/products.

The microfluidic device may include, without limitation, any appropriatenumber of reaction chambers and cavities corresponding to anyappropriate number of reagents to be combined. For example, anotherformat of chambers may include 12×96 (1152 reaction chambers) or 12×32(384 reaction chambers) arrays of chambers and cavities. It should beunderstood that this device can be designed to include any desirednumber of chambers and cavities, corresponding to any desired number ofDNA samples and any solution, as the present disclosure is not limitedin this respect.

Each chamber and each cavity corresponding to the chamber may define anysuitable volume. For example, the volume of chambers of microfluidicdevices described may be less than about 10 microliters, less than about1 microliter, less than about 500 nanoliters, less than about 200nanoliters (e.g., about 150 nanoliters), less than about 100 nanoliters,less than about 50 nanoliters, less than about 20 nanoliters, less thanabout 10 nanoliters, less than 5 nanoliters, less than about 1nanoliter, or any other appropriate volume. The volume defined bycavities corresponding to a particular chamber may also vary.Accordingly, the volume of corresponding cavities of a chamber may beabout equal or may substantially differ.

In some embodiments, the microfluidic device provides for the ability tocombine three or more reagents instead of two. Accordingly, themicrofluidic device may be constructed into an array of chambers whereeach chamber includes three or more corresponding cavities. Each of thecorresponding cavities may be filled, subject to fluid isolation fromneighboring cavities, and the corresponding cavities which werepreviously separated from one another may be fluidly connected so as toallow for mixing of the contents within.

Examples of devices that are able to combine three different reagentswithin a reaction chamber are shown in FIGS. 11A-12B. Though, it can beappreciated that microfluidic devices in accordance with the presentdisclosure may be configured and arranged to mix together any suitablenumber of distinct reagents in different combinations.

FIGS. 11A-11B depict an embodiment of a microfluidic device thatfacilitates the combination of three different reagents in each reactionchamber. Here, the device includes a first layer 200, a second layer 210and a third layer 220. Shown in FIG. 11B, intermediate layers 250, 260are provided on either side of the second layer 210; the firstintermediate layer 250 is positioned between first layer 200 and secondlayer 210, and the second intermediate layer 260 is positioned betweensecond layer 210 and third layer 220.

In this embodiment, chambers in which reactions are contemplated tooccur are formed by three corresponding cavities across three layerswhere each of the layers and, hence, cavities are separated byrespective separating materials prior to being fluidly connected.

As shown, the first layer 200 is formed to include a number of inlets 20for filling cavities 202 that are in fluid connection via channels 204.Each inlet with cavities 202 and channels 204 is also connected to avent 22 to accommodate potential overflow of fluid ingredient and forexhausting excess gas.

Also, the first layer 200 and second layer 210 include inlets 30 andvents 32 for appropriate supply of cavities 212 which are in fluidcommunication via channels 214. The inlets 30 extend through the firstlayer 200 to the second layer 210 so that appropriate fluid ingredientsmay be added at the surface of the first layer 200 from fluid sourcereservoirs and flow into the cavities and channels of the second layer210.

Further, the first layer 200, second layer 210 and third layer 220include inlets 40 and vents 42 for appropriate supply of cavities 222which are in fluid communication via channels 224. The inlets 40 extendthrough the first layer 200 through the second layer 210 and to thethird layer 220 so that appropriate fluid ingredients may be added atthe surface of the first layer 200 from fluid source reservoirs and flowinto the cavities of the third layer 220.

The cross-sectional view of FIG. 11B shows a reaction chamber 201,indicated by the dashed circled region, having corresponding cavities202, 212, 222 that are initially separated by layers 250, 260. Inoperation, fluid ingredient is provided through inlet 20 to the cavities202 of the first layer 200. Further fluid ingredient is provided throughinlet 30 to the cavities 212 of the second layer 210. Additional fluidingredient is provided through inlet 40 to the cavities 222 of the thirdlayer 220. Once the cavities and channels of each of the layers areappropriately supplied with respective fluid ingredients, the channelsof each layer are sealed via any suitable method (e.g., methodsdescribed above involving a sealing material), resulting in fluidisolation of the cavities. Then, openings are formed in the separatingmaterial of the layers 250, 260 that initially had divided correspondingcavities 202, 212, 222. Such openings allow the contents within thosecavities corresponding to the chamber 201 to mix.

FIGS. 12A-12B depict another embodiment of a microfluidic device that isable to combine three different reagents in a reaction chamber. In thisembodiment, an intermediate layer 350 including a separating/sealingmaterial is disposed between a first layer 300 and a second layer 310.Similar to the embodiment shown in FIGS. 11A-11B, chambers in whichreactions are contemplated to occur are formed by three correspondingcavities. However, one of the corresponding cavities of each chamber isdefined by the first layer 300 and the other two cavities are defined bythe second layer 310. The intermediate layer 350 is used to separatecavities corresponding to a chamber, so only one intermediate layer(having a separating/sealing material) and two layers (e.g., layers thatare stiffer) disposed on either side are used for the three-cavitychambers, as opposed to the two intermediate layers and the three layers(e.g., layers that are stiffer) in an alternating arrangement, asprovided in the embodiment of FIGS. 11A-11B.

As shown, the cavities 302 of the first layer 300 are shown to be largerin volume than the cavities 312, 322 of the second layer 310. It can beappreciated that the shape (e.g., depth, contour, etc.) of the cavities302, 312 and 322 may be appropriately determined so as to allow for anysuitable ratio of combination of the different fluid ingredients. Anumber of inlets 20 are provided for filling cavities 302 via channels304, with associated vents 22 to accommodate possible overflow of fluidingredient and excess gas. The second layer 310 includes inlets 30 andvents 32 that are arranged to supply cavities 312, which form a fluidconnection with channels 314, with fluid ingredient. The second layer310 also includes inlets 40 and vents 42 which are arranged to supplycavities 316 via channels 318 with fluid ingredient.

FIG. 12B shows a cross-sectional view of the device of FIG. 12A, whereone of the reaction chambers 301 is indicated by the dashed circledregion. The chamber has corresponding cavities 302, 312, 316 which areall initially separated by the separating material of layer 350. In use,fluid ingredient is provided through inlet 20 to the cavities 302 of thefirst layer 300. Further fluid ingredient is provided through inlet 30to the cavities 312 of the second layer 310. Additional fluid ingredientis provided through inlet 40 to the cavities 316 of the second layer310.

When the cavities and channels of each of the layers are appropriatelyfilled with respective fluid ingredients, the channels of each layer aresealed via any suitable method, resulting in fluid isolation of thecavities. Then, openings are formed in portions of the separatingmaterial that divides corresponding cavities 302, 312, 316 from oneanother so as to allow contents within all three of the cavities to mix.

It can be appreciated that embodiments of the present disclosure are notmeant to be limiting in their particular configuration or arrangement.Accordingly, any suitable number of groups of reagents can be combined,as the present disclosure is not limited in this regard.

In some embodiments, channels connecting adjacent cavities are routed insuch a manner so as to limit the area occupied by the channels on thedevice. For example, channels may be arranged to incorporate multiplebends, allowing for fine control of the distance of channels and betweencavities. Control over the distance between channels may assist incontrolling bond quality between layers of the device, for example, byproviding room for material (e.g., sealing material, fluid, etc.) to bedisplaced such that the displaced material does not interfere with othercomponents/features of the device.

In some embodiments, layers defining channels that are spaced closertogether may exhibit a greater bond quality between layers as comparedwith layers defining channels that are spaced farther apart, forexample, due to potential overflow of material from the channels uponcompression. That is, channels that are spaced so as to take up moresealing material (e.g., closer together) upon compression may providefor generally even bonding between layers while channels spaced furtherapart may take up less sealing material making the sealing material moreprone to bunching, hence, decreasing overall bond quality. As discussedfurther below, the device may include additional space(s) (e.g., reliefchambers, additional cavities) that provide room into which excessmaterials (e.g., fluid ingredient, sealing material, air, etc.) mayenter.

In some embodiments, a number of cavities of the device are connected toa relief chamber which functions to reduce the pressure of fluid insidethe cavities, particularly if and when the device is compressed.

FIG. 13A depicts a number of relief chambers 106, in which reactions(e.g., PCR) are not intended to occur, each chamber 106 connected to acavity 102 via a channel 108. Each relief chamber 106 has a compressiblevolume of air that remains within the relief chamber as liquid fillsinto the cavities. In some embodiments, the relief chamber 106 has onlyone channel for entry and exit of fluid, substantially preventing thecompressible volume of air contained within the chamber from escaping,i.e., the relief chamber is effectively a dead end. Accordingly, thecompressible volume of air within relief chambers provides a built-inmechanism that discourages filling of the relief chamber(s) with fluidingredient while cavities 102 are initially being filled, yet alsoprovides overflow space for the fluid ingredient from the cavities 102upon compression of the device.

In embodiments where the layers of the device are compressed relative toone another so that channels of the device are sealed with a sealingmaterial, the volume inside the cavities may be reduced, leaving lessoccupancy space for fluid within the cavities. For a cavity 102connected to a relief chamber 106 and filled with liquid ingredient,during compression, an excess volume of liquid may overflow from thecavity 102 into the relief chamber 106. The ability for the excessvolume of liquid to overflow from the cavity into the relief chambercompresses the air located within the relief chamber but also allows forthe reduction of pressure and/or resistance buildup within the device tooccur.

In some embodiments, the depth of the channels may affect whether a sealis properly formed within a channel that otherwise fluidly connectsneighboring cavities. When the device is subject to an externallyapplied compressive stress, a shallow channel is more likely to resultin a properly formed seal that prevents fluid flow therein as comparedto a deeper channel. That is, comparatively less deformation (e.g.,provided by applied compression) of the sealing material is required tocreate a suitable seal in a smaller space, allowing for a fine degree ofchannel sealing control. Accordingly, the depth of the channels may bedesigned so as to promote, or discourage, sealing.

In some embodiments, channels that connect cavities to other cavitiesmay be more shallow in depth than channels that are closer to fluidsource reservoirs or those that connect cavities with pressure reliefchambers. Accordingly, channels that connect cavities to one another maybe more easily sealed than channels that are connected to other featuresof the device.

In some embodiments, the channel depth may vary along the same channel.For example, a channel connecting the first cavity along a channel witha fluid source reservoir in a subset of cavities may require anespecially tight seal to be formed near the cavity but such a tight sealmight not be required closer to the fluid source reservoir. As a result,the region of the channel nearest to the cavity may be more shallow thanthe region of the channel nearest to the fluid source reservoir. Or, insome embodiments, deeper regions of a channel may remain substantiallyunfilled by a sealing material upon compression of the device, andshallower areas may become obstructed and filled by the sealing materialupon compression of the device.

Channels which require sealing may have a sufficient amount of a sealingmaterial located around or in otherwise close proximity to the channelsso that a sufficient amount of the sealing material may be deformed intothe channels during sealing actuation (e.g., compression and/orheating). Accordingly, sealing channels, such as those that fluidlyconnect cavities with each other, may be constructed to be longer orfurther away from other features (e.g., fluid source reservoirs, reliefchambers) so that more sealing material is available to deform into thechannels. And for channels where sealing is not preferable, such asthose that connect cavities with relief chambers, the channels may berelatively short and deep so that sealing of these channels does notoccur as readily.

In some embodiments, the device may be formed to include additionalcavities, or pockets, at appropriate locations that are arranged tocollect excess fluid and/or sealing material that may be displaced fromvarious cavities or channels during heating and/or compression of thedevice. Such additional cavities are generally not intended to house areaction, and may be connected to vents so as to remain unpressurizedafter steps of compression/heating of the device. The additionalcavities may be located at regions of the device that would otherwisebecome partially or completely filled with sealing material, forexample, cavities or channels at the edge of an array where overflow maybe more likely to occur.

FIG. 13B shows additional cavities 103, 105 of the device that may besuitable for collecting excess sealing material. For example, theadditional cavity 103 may collect excess sealing material that overflowsresulting from compression of the region between an edge of the deviceand the cavity. The additional cavities 105 located between fluid inputreservoirs may be suitable to collect excess sealing material resultingfrom compression of the region between a cavity and the fluid inputreservoirs, or vents.

In some embodiments, interfaces of the device may provide for a smoothfluid flow transition between channels and cavities. For instance, FIG.14 shows gradual interface 107 between channels and cavities that arerounded which allows fluid to flow evenly between channels and cavities.Otherwise, sharp corners between channels and cavities may result in anoverall increase in fluid flow resistance within the device, forexample, due to capillary resistance from liquid surface tension, orturbulent fluid flow.

In some cases, smooth interfaces between channels and cavities mayreduce the overall pressure required for fluid to fill the cavities.What is more, smooth interfaces between channels and cavities may allowfor channels to be more easily filled with sealing material than wouldotherwise arise from a sharper interface.

In some embodiments, the device may include one or more protrusions,such as ribs, inserts, bumps, ridges, etc., that effectively reduce theamount of compressive force that would otherwise be required to seal theappropriate channels. Such protrusions may, for example, be disposed inalignment with the channels so that, upon application of compressiveforce, compressive stresses are concentrated at channel regions so as tobetter facilitate sealing of the channels.

This stress concentration may promote deformation of the sealingmaterial at the appropriate region(s) resulting in the sealing materialbeing forced into the channels. That is, the protrusions may reduce theoverall compressive force required for the cavities to be sealed. Duringcompression, this arrangement may also result in a smaller amount ofcompressive stresses in regions other than channels that would otherwisebe present, such as over cavity regions. Accordingly, by mitigatingcompressive stress concentrations in regions such as over cavityregions, the overall amount of fluid overflow/displacement to otherareas of the device due to compression is reduced.

In some embodiments, such protrusions may be integral with the sealingmaterial or in contact with the sealing material. Alternatively, theprotrusions may be part of one or more layers of the device. Or, theprotrusions may be part of an external device/component that may be usedto compress layers of the device together. For example, the protrusionsmay be included as part of a thermocycler assembly, or a separate layeraltogether that is used primarily for deforming the sealing material.

As discussed above, once channels of the microfluidic device are sealedsuch that cavities are isolated and the contents of correspondingcavities of each chamber are allowed to mix, a reaction process may beinitiated. In some cases, once reagents are combined, the reactionimmediately takes place. In other cases, a thermal cycling sequenceoccurs to initiate various parts of the reaction, such as in the case ofPCR.

Once the desired reactions are completed, the results are collected. Onepreferred method of collecting the results is through opticaltechniques. Optical collection may be performed using any suitablemethod, such as through absorbance, fluorescence and/or luminescence ofthe contents of the reaction chambers. Or, each chamber may be imagedvia microscopy and an operator may manually observe specific featuresthat result from the reactions within the chambers.

In the context of PCR, once the reagents are combined, the microfluidicdevice would be subject to thermal cycling. In some cases, whenappropriate, the thermal cycler may be the same device that providesheat and/or compressive pressure to the device for sealing the channelsand deforming (e.g., tearing) the separating/sealing material(s).

Accordingly, a user could fill the device with the appropriate reagents,the steps of cavity isolation and combining fluid ingredients togetherthat were initially separated may be performed (manually orautomatically), and the device with combined reagents may be subject tothermal cycling (immediately or after a certain period of time).

In some embodiments, the thermal cycler would provide clamping of thelayers of the microfluidic device together so as to result in goodthermal contact and, hence, reliably quick, responsive temperatureadjustments. Such clamping may also provide for a suitable amount ofexternally applied compression, for example, to seal off the channelsbetween cavities.

The embodiment illustrated in FIG. 15 depicts a microfluidic device 10placed between clamping plates 600, 610 of a thermal cycler. Below plate610, is a thermoelectric element 620 and a heat sink 630, providingtemperature control to the system.

In some embodiments, to seal the channels, the thermal cycler providesfor compressive force applied by clamping plates 600, 610 and/or anincrease in temperature of the device 10, or portions of the device.Though, in some embodiments, at this stage, the temperature would not behigh enough to cause tearing of the separating material. Once thechannels are sealed, the thermal cycler may increase the temperaturefurther to cause the formation of openings in the separating material.Once openings are formed in the separating material, reagents containedwithin the corresponding cavities of each chamber are combined. Once thereagents are suitably combined, the device is subject to thermal cyclingappropriate for PCR to proceed.

In some cases, thermal cycling is preceded by a period of time (e.g.,between 1 and 15 minutes) of high temperature exposure so as to activatethe PCR enzyme. The reagents may then be cycled between two or moretemperature ranges appropriate for PCR. Typically the amount of targetDNA is doubled at the conclusion of each cycle.

PCR detection may be implemented in a number of ways. For instance, inend point PCR, the device undergoes a number of thermal cycles, andafter thermal cycling is complete, the fluorescence of each reactionchamber is measured. In real time PCR, the fluorescence of each chamberis measured periodically during or after cycles, allowing for the amountof DNA in each chamber to be quantified in real time.

For end point PCR detection, the optical detection assembly can becompletely separate from the thermal cycling assembly. Thus, the devicecan be moved from the thermal cycler to the optical detection assemblyafter thermal cycling is complete.

For real time PCR detection, an optical detection assembly may becombined with the thermal cycling assembly allowing for the device to beanalyzed without having to be removed from the thermal cycler. In someembodiments, the clamping plate of the thermal cycler may be translucentto allow for reactions to be optically measured without having to removethe clamp.

In some embodiments, microfluidic devices described herein may be usedfor digital PCR where the amount of DNA in a sample is quantified. Asdiscussed above, in digital PCR, a DNA sample is divided into a largenumber of separate aliquots. Once the sample volume is divided into theseveral aliquots, it is amplified through PCR, and the amount of DNAfrom the original sample can then be calculated by counting the numberof aliquots that have undergone exponential growth through PCR. Theanalysis for digital PCR is a binary process where an aliquot eitherundergoes PCR because at least one molecule of target DNA was presentwithin the aliquot, or the aliquot does not undergo PCR because notarget DNA was present within the aliquot. Thus, the originalconcentration of DNA (e.g., prior to dilution) may be determined basedon a calculated distribution (e.g., Poisson distribution) of positiveand negative aliquots.

For digital PCR, the samples share the same master mix and primersolutions, as the goal for digital PCR is to use a large array ofaliquots into which sample volumes of DNA are provided, to determine howmany molecules of DNA were present in an original sample. Accordingly,it is not necessary to combine different primer solutions with eachsample, as would often be the case in non-digital PCR.

In an embodiment of a microfluidic device that may be used for digitalPCR, shown in FIGS. 16A-16C, the microfluidic device 10 includes a firstlayer 100, a second layer 110 and an intermediate layer 150 sandwichedbetween the layers. The first layer 100 includes an array of cavities102, channels 104, inlets 20 and vents 22. In this embodiment, eachreaction chamber includes a single cavity, as the second layer 110 is amaterial without any channels or cavities defined therein. While notmeant to be limiting, the first and/or second layer(s) may include amaterial that is stiffer (e.g., relatively rigid in comparison) than thematerial of the intermediate layer. Alternatively, in some embodiments,the material of the intermediate layer is stiffer than the materialmaking up the first and/or second layers. The intermediate layer 150includes a sealing material that is adjacent to each of the channels 104of the first layer 100.

While not required, the cavities 102 and channels 104 are furtherdivided into subsets that are each fluidly connected to a respectiveinlet 20. Accordingly, the same fluid ingredient or different fluidingredients may be supplied to each inlet and the cavities associatedwith each inlet. In some embodiments, a microfluidic device suitable fordigital PCR may include single inlet that supplies a fluid ingredient toall cavities of the device through an appropriate network of channelsinterconnected with one another and with the cavities.

The sealing material is arranged to deform into the channels uponappropriate actuation (e.g., compression, heating, etc.) according toany suitable manner so as to provide fluid isolation between thecavities 102. As shown in FIG. 16B, prior to appropriate actuation ofthe device, the channels 104 permit fluid flow between cavities 102.Though, after actuation, the channels 104 are sealed causing thecavities 102 to be fluidly isolated, as shown in FIG. 16C. One of skillin the art would appreciate that any suitable method may be used tocause fluid isolation of the cavities, for example, methods describedand illustrated with respect to FIGS. 9B-9D, or other techniques notexplicitly shown.

Another embodiment of a microfluidic device that may be used for digitalPCR is shown in FIGS. 17A-17C. The microfluidic device includes a firstlayer 100 and a second layer 110. The first layer 100 includes an arrayof cavities 102, channels 104, inlets 20 and vents 22. The second layer110 in this embodiment is similar to the intermediate layer 150 shown inFIGS. 16A-16C in that the second layer includes a sealing material thatsurrounds each of the channels 104 of the first layer 100.

As shown in FIG. 17B, prior to actuation (e.g., compression and/orheating) of the device, the channels 104 permit fluid flow betweencavities 102. Though, after actuation, the channels 104 are sealedcausing the cavities 102 to be fluidly isolated, as shown in FIG. 17C.

In some embodiments, the sealing material of the second layer 150 mayinclude an adhesive material disposed on a substrate (e.g., plasticbacking). Alternatively, this layer may be disposed on the first layer100, or may be composed of a continuous membrane.

It can be appreciated that for embodiments in accordance with thepresent disclosure, the sealing material is not required to be acontinuous monolithic layer, nor is it necessary for it to be of thesame external dimensions as the first layer in which the cavities aredefined. It can be appreciated that the layer(s) of the device,including the sealing material, are not required to have dimensionswhere the layer(s) come(s) into alignment. Nor is it required that thelayer defining the cavities be above or below the sealing material.

Thus, in this embodiment, after the cavities are filled with a suitableDNA sample solution, the layer 150 is subject to heating and/orcompression. It can be appreciated that the sealing material is notrequired to be a continuous layer, as the sealing material may include anumber of separate components.

As discussed above, microfluidic devices described herein may include,or may otherwise engage with another device/component with, protrusionsthat enhance the effects of compression at selected locations.Protrusions may be structural projections that extend/jut outwardly froma layer. In some embodiments, protrusions may have at least onedimension that extends along the plane parallel to the layer that isless than the respective dimension of the cavities of the device. Forexample, the ratio of the width w_(c) of a cavity to the width w_(p) ofa protrusion may be between 1 and 8, between 1.5 and 5, or between 2 and4.

In some embodiments, protrusions may be provided in alignment withparticular channel regions so that when the overall device iscompressed, the protrusions provide a greater amount of stress to thechannel regions as compared with other regions that are not aligned withthe protrusions. While the inclusion of protrusions in certain regionsmay provide for a greater degree of compressive stress to those regions(e.g., regions of channels to be sealed off), the absence of protrusionsin other regions may result in an effective reduction of compressivestress that would otherwise occur over regions where reactions areintended to occur (e.g., cavities, chambers). Mitigating the compressivestress at regions where reactions are intended to occur may bebeneficial to reduce the overall amount of fluid displaced bycompression, and further reduces the overall pressure acting on thesealed regions.

FIGS. 18A-18B depict an embodiment of a microfluidic device 10 similarto that shown in FIGS. 1-4, yet includes an additional layer 160 thathas protrusions 162 that provide for enhanced compression at certainchannel regions of the device. As shown in FIG. 18B, the protrusions 162are aligned with channels 114 of the second layer 110 and channels 104of the first layer 100, such that upon compression of the layers of thedevice 10 together, hence pressing of the protrusions toward thechannels 104, 114, the channels are subject to a greater degree ofcompressive stress than, for example, the cavities 102, 112.Accordingly, the protrusions allow the channels 104, 114 to be moreeasily sealed, i.e., less externally applied compressive force isnecessary for the channels 104, 114 to be sealed off than wouldotherwise be required if the aligned protrusions were not present. Inaddition, the width w_(p) of the protrusions is shown to be less thanthe width w_(c) of the cavities.

Such protrusions (e.g., ribs, bumps, ridges, patterned projections,etc.) for enhancing compressive stresses may be patterned in anysuitable manner. As discussed above, the protrusions may be part of themicrofluidic device itself, a separate thermal cycling apparatus, and/ormay be a separate layer/component that is used primarily forcompression. Alternatively, appropriate protrusions may be formed on thesurface of any of the layers of the device (e.g., on aseparating/sealing material, on appropriate regions of a layer definingcavities and/or channels, etc.).

FIGS. 19A-19B show an embodiment of a microfluidic device 10 similar tothat illustrated in FIGS. 16A-16C, yet includes an additional layer 160that has protrusions 162 that provide for enhanced compression atchannel regions of the device.

FIGS. 20A-20B depict an embodiment of a microfluidic device 10 similarto that illustrated in FIGS. 17A-17C, though, the additional layer 160having protrusions 162 that provide for enhanced compression is alsoincluded. Here, during compression, the protrusions 162 are placed indirect contact with the sealing material of the layer 150.

In some embodiments, a sealing material may be selectively compressed soas to create a number of reaction cavities. For example, channelsdefined by a layer may be filled with a fluid ingredient and a sealingmaterial may be actuated in a manner that results in partitioning of thechannel into reaction cavities that are fluidly isolated from oneanother.

In an embodiment shown in FIG. 21, reaction cavities may be formed bydeforming the sealing material of layer 150 into the channels 104defined by first layer 100 at intervals along the channel; actuated bycompression, heat or a combination of heat and compression of thedevice. The additional layer 160, which may, for example, be a part ofthe device, part of an external compression system, or part of a thermalcycling device on which the microfluidic device is heated, contains anarray of protrusions 162 which partition the channels into suitablelengths, creating cavities corresponding to the desired volume(s) offluid ingredient. In some embodiments, such protrusions 162 may beadapted to jut into the channel with the sealing material. Or, theprotrusions may remain outside the space initially defined by thechannel while suitably deforming the sealing material into thechannel(s). Alternatively, such protrusions may be formed on the sealinglayer 150, or the first layer 100.

As discussed further above, the sealing material may include anysuitable composition. In some embodiments, the sealing material may bean adhesive, e.g., a hot melt adhesive, acrylic adhesive, or otherappropriate adhesive material, positioned on a plastic backing.Alternatively, the sealing material may be a wax, elastomer, or otherrelatively deformable material.

Such a sealing material may be provided as a standard adhesive tape(e.g., packing tape), allowing for relatively simple device assembly.Accordingly, for some embodiments, a molded first layer may define anumber of channels (with optional cavities), input wells, and ventholes. A piece of tape may be attached to the molded layer, covering thechannels (and optional cavities) and the device would essentially beready for use.

The embodiment of the microfluidic device 10 shown in FIGS. 22A-22C issimilar to that of FIG. 21 except that the additional layer 160 includesprotrusions 162 which are formed as elongated ridges. The elongatedridges are provided to control selective compression of the sealingmaterial and are arranged to extend substantially perpendicular to thechannels 104. Accordingly, when the device is subject to compression,the ridges act to concentrate the compressive force in a manner thatcauses deformation of the sealing material into the channels so as todefine an array of reaction cavities in fluid isolation from oneanother, as well as any fluid source reservoir(s).

FIG. 22B shows the device prior to compression where fluid is permittedto flow throughout the channel 104 and cavities are not yet formed. Thatis, in such an embodiment, pre-formed cavities, prior to compression ofthe sealing material, are absent. Upon compression, the protrusions 162are pressed into the sealing material of layer 150 resulting inpartitioning of the channel into cavities 102, shown in FIG. 22C. Theresulting cavities 102 are in fluid isolation, providing for individualreactions (e.g., PCR) to occur in each. As shown, while not required,the width w_(p) of the protrusions is less than the width w_(c) of theresulting cavities.

The resulting cavities in which reactions are able to occur separatefrom other cavities may define any suitable length corresponding to anydesired volume of fluid ingredient. As a result, the use of protrusionsto form separate cavities in which reactions may occur is advantageousin allowing for simple manufacturing and assembly of a microfluidicdevice 10 that does not require substantial effort in aligning variouscomponents/layers of the device.

Accordingly, a suitable sealing material may be deformed into the spaceof the channels through which fluid would otherwise be able to flow andmay further result in appropriate partitioning of the channel intocavities within which separate reactions are able to occur. Thisdeformation may be plastic, hence, permanent in nature. Thus, absentdisassembly of the microfluidic device to remove the sealing materialfrom the channels, such channel sealing is irreversible.

It can be appreciated that various aspects of sealing described hereinare applicable for different applications, such as digital andnon-digital PCR, and/or partitioning of channels into separate cavities.As shown in FIG. 23, the microfluidic device may also be compressedthrough the use of a roller. For example, a roller 170 may be providedso as to travel along the length of the channel(s) 104 (e.g., shown bythe direction indicated by the dashed arrows) resulting in serialdeformation of the sealing material and sealing of the channels 104 andcavities 102 connected by the channels. This method may be advantageousin that the roller would effectively function to progressively displacefluid from each cavity or channel being compressed into the nextuncompressed cavity or out through the vent.

As illustrated in FIG. 23, as the roller 170 moves along and compressesthe channel 104, excess material (e.g., fluid ingredient, air) thatflows from the channel 104 and cavities 102 is displaced to neighboringcavities and channels until the excess material is vented or containedin an appropriate additional cavity and/or relief chamber. Thissequential displacement would reduce the overall pressure that wouldotherwise arise in the cavities and channels due to the compression ofthe fluid ingredient, and would therefore result in stronger sealing ofthe channels.

Devices of the present disclosure may be useful for digital PCR and/ornon-digital PCR. While devices illustrated by the figures are depictedto have relatively few cavities/aliquots, it can be appreciated that anyappropriate number of cavities/aliquots may be implemented. For example,devices used for digital PCR may form 100-10,000 aliquots per sampleinput. In general, the greater the number of partitions for a digitalPCR application, the larger the dynamic range and, hence, the betterstatistical precision of quantification of the sample.

The microfluidic device may provide any suitable volume for eachcavity/aliquot. In some embodiments, a suitable range of volume perpartition (e.g., forming reaction cavities) may be between 10 picolitersand 300 nanoliters, between 500 picoliters and 200 nanoliters, orbetween 1 nanoliter and 100 nanoliters. With the ability to produce alarge number of aliquots, the volume of each individual aliquot can belowered so as to reduce the amount of reagents needed and, hence, theoverall cost.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modification, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

What is claimed is:
 1. A microfluidic device, comprising: a first layerdefining a first cavity; a first channel arranged to provide fluid entryto the first cavity; a second layer defining a second cavity; a secondchannel arranged to provide fluid entry to the second cavity; and atleast one separating material located between the first layer and thesecond layer and providing fluid separation between the first cavity andthe second cavity, the at least one separating material comprising atleast one of plastic, polymer, polyethylene, ethylene-vinyl acetate,polycarbonate, PTFE, polypropylene, elastomer, silicone, siliconerubber, wax, paraffin wax and microcrystalline wax, wherein compressionof the first and second layers relative to one another causes sealing ofthe first channel and the second channel resulting in obstruction of thefirst cavity and the second cavity from further fluid entry, and whereinmanipulation of the at least one separating material causes removal ofthe fluid separation allowing for fluid communication between the firstcavity and second cavity.
 2. The microfluidic device of claim 1, whereinthe first layer defines a plurality of first cavities and the secondlayer defines a plurality of second cavities, the plurality of first andsecond cavities corresponding to an array of chambers, and wherein eachchamber is adapted to house a reaction upon removal of the fluidseparation according to a combination of fluid ingredients provided by aplurality of fluid source reservoirs.
 3. The microfluidic device ofclaim 1, wherein compression of the first and second layers relative toone another causes fluid isolation of the first and second cavities fromadjacent first and second cavities.
 4. The microfluidic device of claim1, wherein compression of the first and second layers relative to oneanother causes substantial filling of the first and second channels witha sealing material.
 5. The microfluidic device of claim 4, wherein theat least one separating material comprises the sealing material.
 6. Themicrofluidic device of claim 5, wherein the at least one separatingmaterial and the sealing material comprise a single deformable film. 7.The microfluidic device of claim 4, wherein the at least one separatingmaterial is independent of the sealing material.
 8. The microfluidicdevice of claim 7, wherein the first layer or the second layer comprisesthe sealing material.
 9. The microfluidic device of claim 4, whereincompression of the first and second layers relative to one anothercauses plastic deformation of the sealing material.
 10. The microfluidicdevice of claim 4, wherein heating of the sealing material promotessubstantial filling of the first and second channels with the sealingmaterial.
 11. The microfluidic device of claim 1, wherein manipulationof the separating material causes irreversible removal of the fluidseparation between the first cavity and second cavity.
 12. Themicrofluidic device of claim 1, wherein tearing of the at least oneseparating material causes removal of the fluid separation between thefirst cavity and second cavity.
 13. The microfluidic device of claim 12,wherein the at least one separating material is in a stressed orstretched state that, upon manipulation, promotes tearing of the atleast one separating material.
 14. The microfluidic device of claim 12,wherein the at least one separating material is in a scored or at leastpartially cut state that, upon manipulation, promotes tearing of the atleast one separating material.
 15. The microfluidic device of claim 1,wherein the at least one separating material is adapted to contractunder application of heat.
 16. The microfluidic device of claim 1,wherein manipulation of the at least one separating material comprisesapplication of heat.
 17. The microfluidic device of claim 1, whereinmanipulation of the at least one separating material comprisesapplication of at least one of a compressive force, a magneticallydriven force, laser irradiation and centrifugation.
 18. The microfluidicdevice of claim 1, further comprising at least one relief cavity fluidlyconnected to a corresponding cavity and adapted to receive an overflowof fluid from the corresponding cavity.
 19. The microfluidic device ofclaim 18, wherein the at least one relief cavity is adapted to remainsubstantially filled with a volume of air upon supply of thecorresponding cavity with fluid.
 20. The microfluidic device of claim 4,further comprising at least one storage cavity adapted to prevent excesssealing material from filling the first cavity or the second cavity. 21.The microfluidic device of claim 1, in combination with a systemarranged to perform polymerase chain reaction.
 22. The microfluidicdevice of claim 1, wherein the first and second channels aresubstantially aligned with a plurality of protrusions extending from atleast one of the first layer, the second layer and an additional layer.23. A microfluidic device, comprising: a first layer defining a firstcavity; a first channel arranged to provide fluid entry to the firstcavity; a second layer defining a second cavity; a second channelarranged to provide fluid entry to the second cavity; and at least oneseparating material providing fluid separation between the first cavityand the second cavity, wherein compression of the first and secondlayers relative to one another causes sealing of the first channel andthe second channel resulting in obstruction of the first cavity and thesecond cavity from further fluid entry, and wherein manipulation of theat least one separating material causes removal of the fluid separationallowing for fluid communication between the first cavity and secondcavity, wherein the at least one separating material comprises at leastone of polycarbonate, PTFE, silicone, polypropylene and paraffin wax.